{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,2]],"date-time":"2026-09-02T15:15:53Z","timestamp":1788362153799,"version":"build-2803163510"},"reference-count":28,"publisher":"MDPI AG","issue":"17","license":[{"start":{"date-parts":[[2026,9,2]],"date-time":"2026-09-02T00:00:00Z","timestamp":1788307200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000062","name":"National Institutes of Health","doi-asserted-by":"publisher","award":["R01 DK132387"],"award-info":[{"award-number":["R01 DK132387"]}],"id":[{"id":"10.13039\/100000062","id-type":"DOI","asserted-by":"publisher"}]},{"award":["R01 DK132387"],"award-info":[{"award-number":["R01 DK132387"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"DOI":"10.13039\/100008871","name":"Advanced Postdoctoral Fellowship","doi-asserted-by":"publisher","award":["3-APF-2024-1492-A-N"],"award-info":[{"award-number":["3-APF-2024-1492-A-N"]}],"id":[{"id":"10.13039\/100008871","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000041","name":"Pathway to Stop Diabetes Initiator Award by ADA","doi-asserted-by":"publisher","award":["1-26-INI-0784"],"award-info":[{"award-number":["1-26-INI-0784"]}],"id":[{"id":"10.13039\/100000041","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Thomas H. Maren Research Excellence Postdoctoral Award by UF"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cells"],"abstract":"<jats:p>Efficient and reproducible generation of functional stem cell-derived \u03b2 cells (sBC) remains a major challenge for basic research and cell replacement therapy, partly due to incomplete understanding of endocrine induction during differentiation and challenges with clinical scale-up. Here, we dissect the individual contributions of commonly used endocrine differentiation factors on pancreatic progenitor maintenance, endocrine commitment, and hormone subset generation in a scalable 3D differentiation system. We demonstrated that starting pluripotent stem cell cluster size is a critical determinant for downstream sBC generation. We also verify that a commonly employed combination of endocrine induction molecules efficiently drives endocrine lineage commitment but yields limited \u03b2-cell generation. Detailed analysis of the effects of individual endocrine induction molecules revealed distinct effects: EGF or KGF preserved NKX6.1+ progenitors without induction of endocrine differentiation; Notch or BMP inhibition robustly induced endocrine marker expression but concurrently reduced NKX6.1 expression, resulting in predominant generation of glucagon-expressing cells; retinoic acid, thyroid hormone (T3), or TGF\u03b2 inhibition maintained high NKX6.1 levels while also promoting efficient insulin+ endocrine differentiation. These findings indicate NKX6.1 protein maintenance as a key determinant of human \u03b2-cell generation and show that endocrine differentiation factors exert divergent effects on lineage progression.<\/jats:p>","DOI":"10.3390\/cells15171600","type":"journal-article","created":{"date-parts":[[2026,9,2]],"date-time":"2026-09-02T14:35:29Z","timestamp":1788359729000},"page":"1600","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Physical and Biochemical Determinants of Endocrine Fate in 3D Suspension Culture Based Stem Cell-Derived \u03b2-Cell Differentiation"],"prefix":"10.3390","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-5873-6473","authenticated-orcid":false,"given":"Roberto","family":"Castro-Gutierrez","sequence":"first","affiliation":[{"name":"Diabetes Institute, University of Florida, Gainesville, FL 32610, USA"},{"name":"Department of Pharmacology and Therapeutics, University of Florida, Gainesville, FL 32610, USA"},{"name":"UCSF Diabetes Center, University of California San Francisco, San Francisco, CA 94134, USA"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"conceptualization","vocabulary":"credit"},{"role":"methodology","vocabulary":"credit"},{"role":"investigation","vocabulary":"credit"},{"role":"visualization","vocabulary":"credit"},{"role":"writing-review-editing","vocabulary":"credit"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5092-5744","authenticated-orcid":false,"given":"Ali H.","family":"Shilleh","sequence":"additional","affiliation":[{"name":"Barbara Davis Center for Diabetes, Department of Pediatrics, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA"},{"name":"Oxford Centre for Diabetes, Endocrinology and Metabolism, Radcliffe Department of Medicine, University of Oxford, Churchill Hospital, Oxford OX3 7LE, UK"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"conceptualization","vocabulary":"credit"},{"role":"methodology","vocabulary":"credit"},{"role":"investigation","vocabulary":"credit"},{"role":"visualization","vocabulary":"credit"},{"role":"writing-review-editing","vocabulary":"credit"}]},{"given":"Jessie M.","family":"Barra","sequence":"additional","affiliation":[{"name":"Diabetes Institute, University of Florida, Gainesville, FL 32610, USA"},{"name":"Department of Pharmacology and Therapeutics, University of Florida, Gainesville, FL 32610, USA"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"writing-original-draft","vocabulary":"credit"},{"role":"methodology","vocabulary":"credit"},{"role":"investigation","vocabulary":"credit"},{"role":"writing-review-editing","vocabulary":"credit"},{"role":"visualization","vocabulary":"credit"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1742-6783","authenticated-orcid":false,"given":"Shane P. M.","family":"Williams","sequence":"additional","affiliation":[{"name":"Barbara Davis Center for Diabetes, Department of Pediatrics, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"methodology","vocabulary":"credit"},{"role":"investigation","vocabulary":"credit"},{"role":"writing-review-editing","vocabulary":"credit"}]},{"given":"Balachandar","family":"Nedumaran","sequence":"additional","affiliation":[{"name":"Barbara Davis Center for Diabetes, Department of Pediatrics, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"methodology","vocabulary":"credit"},{"role":"investigation","vocabulary":"credit"},{"role":"writing-review-editing","vocabulary":"credit"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4796-6542","authenticated-orcid":false,"given":"Taylor M.","family":"Triolo","sequence":"additional","affiliation":[{"name":"Barbara Davis Center for Diabetes, Department of Pediatrics, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"methodology","vocabulary":"credit"},{"role":"investigation","vocabulary":"credit"},{"role":"writing-review-editing","vocabulary":"credit"}]},{"given":"Matthias","family":"Hebrok","sequence":"additional","affiliation":[{"name":"Center for Organoid Systems, University Hospital rechts der Isar (MRI), Technical University Munich, 85748 Garching, Germany"},{"name":"Institute for Diabetes Organoid Technology, Helmholtz Munich, Helmholtz Diabetes Center, Ingolst\u00e4dter Landstra\u00dfe 1, 85764 Neuherberg, Germany"},{"name":"Munich Institute of Biomedical Engineering (MIBE), Technical University of Munich, 85748 Garching, Germany"},{"name":"German Center for Diabetes Research (DZD), 85764 Neuherberg, Germany"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"writing-review-editing","vocabulary":"credit"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5117-2927","authenticated-orcid":false,"given":"Holger A.","family":"Russ","sequence":"additional","affiliation":[{"name":"Diabetes Institute, University of Florida, Gainesville, FL 32610, USA"},{"name":"Department of Pharmacology and Therapeutics, University of Florida, Gainesville, FL 32610, USA"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"conceptualization","vocabulary":"credit"},{"role":"methodology","vocabulary":"credit"},{"role":"investigation","vocabulary":"credit"},{"role":"supervision","vocabulary":"credit"},{"role":"funding-acquisition","vocabulary":"credit"},{"role":"project-administration","vocabulary":"credit"},{"role":"writing-original-draft","vocabulary":"credit"},{"role":"writing-review-editing","vocabulary":"credit"},{"role":"visualization","vocabulary":"credit"}]}],"member":"1968","published-online":{"date-parts":[[2026,9,2]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"428","DOI":"10.1016\/j.cell.2014.09.040","article-title":"Generation of functional human pancreatic \u03b2 cells in vitro","volume":"159","author":"Pagliuca","year":"2014","journal-title":"Cell"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1121","DOI":"10.1038\/nbt.3033","article-title":"Reversal of diabetes with insulin-producing cells derived in vitro from human pluripotent stem cells","volume":"32","author":"Rezania","year":"2014","journal-title":"Nat. Biotechnol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"2432","DOI":"10.1002\/stem.1489","article-title":"Enrichment of human embryonic stem cell-derived NKX6.1-expressing pancreatic progenitor cells accelerates the maturation of insulin-secreting cells in vivo","volume":"31","author":"Rezania","year":"2013","journal-title":"Stem Cells"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"351","DOI":"10.1016\/j.stemcr.2018.12.012","article-title":"Acquisition of dynamic function in human stem cell-derived \u03b2 cells","volume":"12","author":"Song","year":"2019","journal-title":"Stem Cell Rep."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1759","DOI":"10.15252\/embj.201591058","article-title":"Controlled induction of human pancreatic progenitors produces functional beta-like cells in vitro","volume":"34","author":"Russ","year":"2015","journal-title":"EMBO J."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"263","DOI":"10.1038\/s41556-018-0271-4","article-title":"Recapitulating endocrine cell clustering in culture promotes maturation of human stem-cell-derived \u03b2 cells","volume":"21","author":"Nair","year":"2019","journal-title":"Nat. Cell Biol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"108067","DOI":"10.1016\/j.celrep.2020.108067","article-title":"Single-Cell Transcriptome Profiling Reveals beta Cell Maturation in Stem Cell-Derived Islets after Transplantation","volume":"32","author":"Augsornworawat","year":"2020","journal-title":"Cell Rep."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"443","DOI":"10.1038\/nbt1393","article-title":"Pancreatic endoderm derived from human embryonic stem cells generates glucose-responsive insulin-secreting cells in vivo","volume":"26","author":"Kroon","year":"2008","journal-title":"Nat. Biotechnol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"2554","DOI":"10.2337\/db20-0873","article-title":"ENTPD3 Marks Mature Stem Cell-Derived beta-Cells Formed by Self-Aggregation In Vitro","volume":"70","author":"Docherty","year":"2021","journal-title":"Diabetes"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"3038","DOI":"10.1073\/pnas.1400709111","article-title":"Differentiated human stem cells resemble fetal, not adult, \u03b2 cells","volume":"111","author":"Hrvatin","year":"2014","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"11463","DOI":"10.1038\/ncomms11463","article-title":"Generation of stem cell-derived \u03b2-cells from patients with type 1 diabetes","volume":"7","author":"Millman","year":"2016","journal-title":"Nat. Commun."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"368","DOI":"10.1038\/s41586-019-1168-5","article-title":"Charting cellular identity during human in vitro \u03b2-cell differentiation","volume":"569","author":"Veres","year":"2019","journal-title":"Nature"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"877","DOI":"10.1038\/23716","article-title":"Notch signalling controls pancreatic cell differentiation","volume":"400","author":"Apelqvist","year":"1999","journal-title":"Nature"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"81","DOI":"10.1146\/annurev-cellbio-101512-122405","article-title":"Pancreas organogenesis: From lineage determination to morphogenesis","volume":"29","author":"Shih","year":"2013","journal-title":"Annu. Rev. Cell Dev. Biol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1948","DOI":"10.2337\/db09-1010","article-title":"Mesenchymal bone morphogenetic protein signaling is required for normal pancreas development","volume":"59","author":"Ravassard","year":"2010","journal-title":"Diabetes"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"3330","DOI":"10.1038\/s41467-021-23525-x","article-title":"Chemical combinations potentiate human pluripotent stem cell-derived 3D pancreatic progenitor clusters toward functional beta cells","volume":"12","author":"Liu","year":"2021","journal-title":"Nat. Commun."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"101477","DOI":"10.1016\/j.molmet.2022.101477","article-title":"Emerging diabetes therapies: Bringing back the beta-cells","volume":"60","author":"Basile","year":"2022","journal-title":"Mol. Metab."},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"de Almeida Fuzeta, M., Bernardes, N., Oliveira, F.D., Costa, A.C., Fernandes-Platzgummer, A., Farinha, J.P., Rodrigues, C.A.V., Jung, S., Tseng, R.J., and Milligan, W. (2020). Scalable Production of Human Mesenchymal Stromal Cell-Derived Extracellular Vesicles Under Serum-\/Xeno-Free Conditions in a Microcarrier-Based Bioreactor Culture System. Front. Cell Dev. Biol., 8.","DOI":"10.3389\/fcell.2020.553444"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"694","DOI":"10.1007\/s00125-011-2379-y","article-title":"INS(GFP\/w) human embryonic stem cells facilitate isolation of in vitro derived insulin-producing cells","volume":"55","author":"Micallef","year":"2012","journal-title":"Diabetologia"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"2016","DOI":"10.2337\/db11-1711","article-title":"Maturation of human embryonic stem cell-derived pancreatic progenitors into functional islets capable of treating pre-existing diabetes in mice","volume":"61","author":"Rezania","year":"2012","journal-title":"Diabetes"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"138","DOI":"10.1016\/j.stemcr.2019.11.010","article-title":"Analysis of Differentiation Protocols Defines a Common Pancreatic Progenitor Molecular Signature and Guides Refinement of Endocrine Differentiation","volume":"14","author":"Jensen","year":"2020","journal-title":"Stem Cell Rep."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1607","DOI":"10.1073\/pnas.97.4.1607","article-title":"neurogenin3 is required for the development of the four endocrine cell lineages of the pancreas","volume":"97","author":"Gradwohl","year":"2000","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"177","DOI":"10.4161\/isl.1.3.9877","article-title":"Neurogenin3: A master regulator of pancreatic islet differentiation and regeneration","volume":"1","author":"Rukstalis","year":"2009","journal-title":"Islets"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"5533","DOI":"10.1242\/dev.127.24.5533","article-title":"Homeobox gene Nkx6.1 lies downstream of Nkx2.2 in the major pathway of beta-cell formation in the pancreas","volume":"127","author":"Sander","year":"2000","journal-title":"Development"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"1262","DOI":"10.1016\/j.celrep.2013.08.010","article-title":"Nkx6.1 is essential for maintaining the functional state of pancreatic beta cells","volume":"4","author":"Taylor","year":"2013","journal-title":"Cell Rep."},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Schaffer, A.E., Taylor, B.L., Benthuysen, J.R., Liu, J., Thorel, F., Yuan, W., Jiao, Y., Kaestner, K.H., Herrera, P.L., and Magnuson, M.A. (2013). Nkx6.1 controls a gene regulatory network required for establishing and maintaining pancreatic Beta cell identity. PLoS Genet., 9.","DOI":"10.1371\/journal.pgen.1003274"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"298","DOI":"10.1038\/s41467-017-00129-y","article-title":"ROCKII inhibition promotes the maturation of human pancreatic beta-like cells","volume":"8","author":"Ghazizadeh","year":"2017","journal-title":"Nat. Commun."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"460","DOI":"10.1038\/s41587-020-0430-6","article-title":"Targeting the cytoskeleton to direct pancreatic differentiation of human pluripotent stem cells","volume":"38","author":"Hogrebe","year":"2020","journal-title":"Nat. Biotechnol."}],"container-title":["Cells"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2073-4409\/15\/17\/1600\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,2]],"date-time":"2026-09-02T14:42:40Z","timestamp":1788360160000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2073-4409\/15\/17\/1600"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,2]]},"references-count":28,"journal-issue":{"issue":"17","published-online":{"date-parts":[[2026,9]]}},"alternative-id":["cells15171600"],"URL":"https:\/\/doi.org\/10.3390\/cells15171600","relation":{},"ISSN":["2073-4409"],"issn-type":[{"value":"2073-4409","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9,2]]}}}